Ivermectin for the treatment of parasites affecting sheep was evaluated in 35
controlled efficacy trials. There were 748 sheep involved, of which 520 were
given ivermectin and 228 served as controls.

Dose-Titration Trials

Dose-titration studies were carried out to determine the dose of
ivermectin given once orally that would control gastrointestinal nematodes,
lungworms, and nasal bots. Nineteen trials were conducted using ll9 control and
373 ivermectin-treated sheep. Dosages of 50 mcg/kg to 400 mcg/kg were employed.
Local procedures were followed regarding animal husbandry during the trials as
well as for allocating, dosing, collecting samples, enumerating and identifying
parasites, and performing necropsies. The parasitisms were artificially induced
in 13 of the trials, naturally acquired in two trials, and induced infections
were superimposed in four trials. Based on the data collected, a minimum
effective dose of 200 mcg ivermectin/kg bodyweight was determined.

Trial 4828 was conducted in the United States to establish the optimum
effective level of ivermectin against nematodes. Sheep were given infective
larvae on days 27 and 7 before treatment. Prior to treatment, 25 sheep were
randomly allocated to four groups. Ten sheep were used as controls and the
other groups had five sheep each. Control sheep were given propylene glycol
vehicle, and ivermectin was given once orally to the sheep in the other groups
at 50 mcg/kg, 100 mcg/kg, or 200 mcg/kg. Necropsies were performed seven days
after treatment, and the reductions listed below were recorded. No adverse
reactions occurred. Data from 15 additional sheep given another drug are
irrelevant to this summary and have been deleted.

Trial 4829 was conducted in the United States to establish the optimum
effective level of ivermectin against nematodes. The sheep were naturally
infected, but were given additional infective larvae 16 days prior to
treatment. On the day before treatment, 25 sheep were randomly allocated to
four groups.
Ten sheep were used as controls and the other groups had five sheep each.
Control sheep were given propylene glycol vehicle and ivermectin was given once
orally to the sheep in the other groups at 50 mcg/kg, 100 mcg/kg, or 200
mcg/kg. Necropsies were performed six days after treatment, and the reductions
listed below were recorded. No adverse reactions occurred. Data from an
additional 15 sheep given another drug are irrelevant to this summary and have
been deleted.

Trial 5748 was conducted in Australia to establish the optimum effective level
of ivermectin against nematodes. Twenty sheep were artificially infected and
randomly allocated to groups of equal size. After the nematodes had reached
adulthood, each sheep was given either propylene glycol vehicle or ivermectin
once orally at 50 mcg/kg, 100 mcg/kg, or 200 mcg/kg.
Necropsies were performed seven to nine days after treatments, and the
reductions (P<0.05) listed below were recorded. No adverse reactions
occurred. Data from an additional 15 sheep given another drug are irrelevant to
this summary and have been deleted.

Trial 5759 was conducted in Australia as a dose-titration trial and to compare
the efficacy of the individual ivermectin components formulated in propylene
glycol against immature nematodes. Forty sheep were artificially infected and
randomly allocated to eight groups of equal size. The sheep in both control
groups were given vehicle, 22,23-dihydroavermectin B1a was given once orally to
the sheep in three groups at 25 mcg/kg, 70 mcg/kg, or 200 mcg/kg, and
22,23-dihydroavermectin B1b was given once orally to the sheep in the other
three groups at
25 mcg/kg, 70 mcg/kg, or 200 mcg/kg. Treatments were administered when the
nematodes were in the fourth larval stage (L4) of development. Necropsies were
performed 13 to 15 days after treatment, and the reductions (P<0.05) listed
below were recorded. No adverse reactions occurred.

Trial 6074 was conducted in South Africa to establish the optimum effective
level of ivermectin against nematodes. Twenty sheep were artificially infected
and randomly allocated to four groups of equal size. When the nematodes were in
the fourth larval stage (L4), each sheep was given propylene glycol vehicle or
ivermectin once orally at 50 mcg/kg, 100 mcg/kg, or 200 mcg/kg. Necropsies were
performed 14 to 17 days after treatment, and the reductions (P<0.05) listed
below were recorded. No adverse reactions occurred. Data from an additional 15
sheep given another drug are irrelevant to this summary and have been
deleted.

Trial 6637 was conducted in the United Kingdom to establish the optimum
effective level of ivermectin in propylene glycol vehicle against immature
nematodes. Twenty-five sheep on a betamethasone regimen were artificially
infected and randomly allocated to five groups of equal size. Control sheep
were given propylene glycol vehicle and ivermectin was given once orally to the
sheep in the other groups at 25 mcg/kg, 50 mcg/kg, 100 mcg/kg, or 200 mcg/kg.
Treatments were administered when the nematodes were in the fourth larval stage
(L4) of development. Necropsies were performed seven days after treatment, and
the reductions (P<0.05)
listed below were recorded. No adverse reactions occurred.

Trial 6639 was conducted in the United Kingdom to establish the optimum
effective level of ivermectin in propylene glycol vehicle against adult
nematodes. Twenty sheep on a betamethasone regimen were artificially infected
and randomly allocated to four groups of equal size. Control sheep were given
propylene glycol vehicle and ivermectin was given once orally to the sheep in
the other groups at 50 mcg/kg, 100 mcg/kg, and 200 mcg/kg. Treatments were
administered when the nematodes had been allowed to develop to the adult stage.
Necropsies were performed ten days after treatment, and the reductions
(P<0.05) listed below were recorded. No adverse reactions occurred.

Trial 6640 was conducted in the United Kingdom to establish the optimum
effective level of ivermectin against nematodes. Twenty-five sheep were
artificially infected and randomly allocated to five groups of equal size. Each
sheep was given propylene glycol vehicle or ivermectin once orally at 25
mcg/kg, 50 mcg/kg, l00 mcg/kg, or 200 mcg/kg when the nematodes were in the
fourth larval stage (L4) of development. Necropsies were performed 12 days
after treatment, and the reductions (P<0.05) listed below were recorded. No
adverse reactions occurred.

Trial 6647 was conducted in Australia to establish the optimum effective level
of ivermectin against nematodes. Twenty sheep were artificially infected and
randomly allocated to five groups of equal size. Each sheep was given propylene
glycol vehicle or ivermectin once orally at 25 mcg/kg, 50 mcg/kg, 100 mcg/kg,
or 200 mcg/kg when the nematodes were in the fourth larval stage (L4) of
development. Necropsies were performed 12 or 13 days after treatment, and the
reductions (P<0.05) listed below were recorded. No adverse reactions
occurred.

Trial 6666 was conducted in Australia to establish the optimum effective level
of ivermectin against nematodes. Twenty sheep naturally infected were randomly
allocated to four groups of equal size. Each sheep was given propylene glycol
vehicle or ivermectin once orally at 100 mcg/kg, 200 mcg/kg, or 400 mcg/kg.
Necropsies were performed six or seven days after treatment, and the reductions
(P<0.05) listed below were recorded. No adverse reactions occurred. Data
from an additional ten sheep given other drugs are irrelevant to this summary
and have been deleted.

Trial 6676 was conducted in Australia to establish the optimum effective level
of ivermectin against immature nematodes, and to compare the responses of two
ivermectin component formulations (i.e., 80% dihydroavermectin B1a and 20% B1b
versus 95% dihydroavermectin B1a and 5% B1b). Forty-five sheep artificially
infected were randomly allocated to nine groups of equal size. Control sheep
were given propylene glycol vehicle and sheep in the other groups were given 50
mcg/kg, 100 mcg/kg, 200 mcg/kg, or 400 mcg/kg of either ivermectin formulation
(80% B1a or 95% B1a). necropsies were performed 12 to 14 days after treatment,
and the reductions (P<0.05) listed below were recorded. The two formulations
were considered to be equivalent. No adverse reactions occurred.

Trial 6731 was conducted in South Africa to establish the optimum effective
level of ivermectin against immature nematodes. Twenty-five sheep were
artificially infected and randomly allocated to five groups of equal size. When
the nematodes were in the fourth larval stage (L4) of development, the sheep
were given either propylene glycol vehicle or ivermectin at 25 mcg/kg, 50
mcg/kg, 100 mcg/kg, or 200 mcg/kg. Necropsies were performed 17 to 19 days
after treatment, and the reductions (P<0.05) listed below were recorded. No
adverse reactions occurred.

Trial 6734 was conducted in South Africa to establish the optimum effective
dose of ivermectin, primarily against the nose bot (Oestrus ovis) but
also against Haemonchus contortus. Twenty-four sheep naturally infected
were randomly allocated to four groups of equal size. The sheep were given
either propylene glycol vehicle or ivermectin at 100 mcg/kg, 200 mcg/kg, or 400
mcg/kg.
Necropsies were performed five days after treatment and the reductions
(P<0.05) listed below were recorded. No adverse reactions occurred.

Trial 7076 was conducted in the United States to establish the optimum
effective level of ivermectin against nematodes. The sheep were naturally
infected, but were given additional larvae 29 days prior to treatment. Twenty
sheep were randomly allocated to four groups of equal size. Control sheep were
given propylene glycol vehicle and ivermectin was given once orally to the
sheep in the other groups at 100 mcg/kg, 200 mcg/kg, or 400 mcg/kg. Necropsies
were performed seven days after treatment and the reductions listed below were
recorded. No adverse reactions occurred.

Trial 7077 was conducted in the United States to establish the optimum
effective level of ivermectin against nematodes. The sheep were naturally
infected, but were given additional larvae seven days prior to treatment.
Twenty sheep were randomly allowed to four groups of equal size. Control sheep
were given propylene glycol vehicle and ivermectin was given once orally to the
sheep in the other groups at 100 mcg/kg, 200 mcg/kg, or 400 mcg/kg. Necropsies
were performed seven days after treatment, and the reductions (P<0.05)
listed below were recorded. No adverse reactions occurred.

Trial 7078 was conducted in the United States to establish the optimum
effective level of ivermectin in propylene glycol vehicle against immature
nematodes. Twenty-four sheep were artificially infected and randomly allocated
to four groups of equal size. Control sheep were given propylene glycol vehicle
and ivermectin was given once orally to the sheep in the other groups at 100
mcg/kg, 200 mcg/kg, or 300 mcg/kg. Treatments were administered when the
nematodes were in the fourth larval stage (L4) of development. Necropsies were
performed 12 days after treatment, and the reductions listed below were
recorded. No adverse reactions occurred.

Trial 7088 was conducted in the United States to establish the optimum
effective level of ivermectin against nematodes. Three weeks after inoculation
with infective larvae, twenty sheep (also naturally infected) were randomly
allocated to five groups of equal size. Control sheep were given propylene
glycol vehicle and ivermectin was given once orally to the sheep in the other
groups at 50 mcg/kg, 100 mcg/kg, 200 mcg/kg, or 400 mcg/kg. necropsies were
performed seven days after treatment, and the reductions listed below were
recorded. No adverse reactions occurred.

Trial 7098 was conducted in the United States to establish the optimum
effective level of ivermectin against the nasal bot, Oestrus ovid.
Twenty-four naturally infected sheep were randomly assigned to five groups.
Four sheep were used as untreated controls, five were given propylene glycol
vehicle, and ivermectin was given to the sheep in the other groups (n=5/group)
at 50 mcg/kg, 100 mcg/kg, or 200 mcg/Kg. Necropsies were performed seven or
eight days after treatment, and the reductions (P<0.05) listed below were
recorded. No adverse reactions occurred.

Trial 7937 was conducted in Australia to establish the optimum effective level
of ivermectin in either propylene glycol or micelle vehicles against immature
nematodes. Fifty sheep were artificially infected and randomly allocated to ten
groups of equal size. Control sheep in one group were given propylene glycol
vehicle, control sheep in another group were given micelle vehicle, and sheep
in the other groups were given ivermectin formulated in propylene glycol
vehicle or in micelle vehicle at 25 mcg/kg, 50 mcg/kg, 100 mcg/kg, or 200
mcg/kg. necropsies were performed 12 to 14 days after treatment, and the
reductions (P<0.05) listed below were recorded. No differences between the
formulations were detected. No adverse reactions occurred.

Ivermectin is defined as a combination of two homologues, including not
less than 80% 22,23-dihydroavermectin B1a and not more than 20%
22,23-dihydroavermecttn B1b (MK-933). Some of the trials early in the
developmental program were conducted with a combination containing not less
than 95% of the B1a homologue (MK-932). The definition of ivermectin includes
both combinations but, nonetheless, a comparison was conducted (trial 6676, p.
15) and the efficacy of each was determined to be equivalent. An additional
comparison (trial 5759, p.7) was made of the efficacy of the two homologues
separately, and each was determined equally efficacious.

The vehicle (propylene glycol) used in the dose-titrations trials caused the
sheep to cough more than was clinically acceptable. Hence, a micelle
formulation was evaluated (trial 7937, p.23) and, with one minor change in
components, was adopted for commercial use following further comparison in two
other trials (7452, p.32; 8451, p.43).

Dose-Confirmation Trials

Confirmation of the dose selected for gastrointestinal and pulmonary nematodes
was accomplished in 16 controlled trials. The confirmation data were obtained
using a total of 147 sheep treated with ivermectin at 200 mcg/kg bodyweight and
109 controls. Infections were acquired naturally in four trials, induced in lO
trials, and superimposed in two trials. All the sheep were killed 7 to 14 days
after treatment for parasite recoveries.

Trial 6665 was conducted in Australia to confirm the efficacy of ivermectin
against nematodes. Ten sheep with natural infections were randomly allocated to
two groups of equal size. Control sheep were untreated and sheep in the other
group were given ivermectin in propylene glycol once orally at 200 mcg/kg.
Necropsies were performed eight days after treatment, and the reductions
(P<0.05) listed below were recorded. No adverse
reactions occurred.

Trial 6699 was conducted in South Africa to confirm the efficacy of ivermectin
against nematodes. Twenty sheep were artificially infected and randomly
allocated to 3 groups (1 indicator, 7 controls and 12 treated with ivermectin).
Control sheep were untreated and sheep in the other group were given ivermectin
in propylene glycol once orally at 200 mcg/kg when the nematodes were in the
third (Haemonchus contortus, Trichostrongylus colubriformis, and
Chabertia ovina) or fourth (Strongyloides papillosus) larval
stage of development. Necropsies were performed on the 34th, 35th and 3?th days
after treatment. The reductions (P<0.05) listed below were recorded. No
adverse reactions occurred.

Trial 6700 was conducted in South Africa to confirm the efficacy of ivermectin
against nematodes. Twenty sheep were artificially infected and randomly
allocated. One group of seven sheep was used as untreated controls, a second
group of 12 sheep was given ivermectin in propylene glycol once orally at 200
mcg/kg, and one sheep was an infectivity indicator control. The nematodes were
in the fourth larval stage (L4) of development on the day of treatment.
Necropsies were performed 23 to 25 days after treatment, and the reductions
(P<0.05) listed below were recorded. No adverse reactions occurred.

Trial 6854 was conducted in Brazil to confirm the efficacy of ivermectin
against Oestrus ovis. Ten sheep with natural infections were randomly
allocated to two groups of equal size. Control sheep were untreated, and sheep
in the other group were given ivermectin in propylene glycol once orally at 200
mcg/kg. Necropsies were performed five days after treatment. Each of the five
control sheep harbored all three larval stages (bors) of
O. ovis at necropsy, whereas none of the ivermectin-treated sheep
harbored any live bots. No adverse reactions occurred. Data from five other
sheep given another drug are irrelevant to this summary and have been
deleted.

Trial 7257 was conducted in the United Kingdom to confirm the efficacy of
ivermectin against Nematodirus battus. Ten sheep were artificially
infected and randomly allocated to two groups of equal size. Control sheep were
given vehicle, and sheep in the other group were given ivermectin in propylene
glycol once orally at 200 mcg/kg. Necropsies were performed seven days after
treatment and the reductions (P<0.05) listed below were recorded. Data from
five other sheep given ivermectin at 100 mcg/kg are irrelevant to this summary
and have been deleted. No adverse reactions occurred.

Trial 7452 was conducted in Australia to compare the efficacy of ivermectin in
propylene glycol or micelle formulations against immature nematodes. Twenty
sheep were artificially infected and randomly allocated to four groups of equal
size. Sheep in two groups received either vehicle, and sheep in the other two
groups received ivermectin at 200 mcg/kg when formulated in either vehicle.
Treatments were administered when the nematodes were in the fourth larval stage
(L4) of development. necropsies were performed seven to nine days after
treatment, and the reductions (P<0.05) listed below were recorded. Based on
the data obtained, ivermectin given in either formulation was concluded to be
equivalent. Data from another ten sheep given a third formulation are
irrelevant to this summary and have been deleted. No adverse reactions
occurred.

Trial 7662 was conducted in New Zealand to confirm the efficacy of
ivermectin against nematodes. Fourteen sheep with natural infections as well as
induced infections of Dictyocaulus filaria and Haemonchus contortus
were randomly allocated to two groups of equal size. Control sheep were
given vehicle, and sheep in the other group were given ivermectin in micelle
solution once orally at 200 mcg/kg. necropsies were performed seven to nine
days after treatment and the reductions (p<0.05) listed below were recorded.
Data from 21 other sheep given other drugs are irrelevant to this summary and
have been deleted. No adverse reactions occurred.

Trial 7683 was conducted in South Africa to confirm the efficacy of ivermectin
against nematodes. Twenty sheep were artificially infected and allocated to two
groups. Eight sheep were untreated controls, and 12 sheep were given ivermectin
in propylene glycol once orally at 200 mcg/kg when the nematodes had reached
the adult stage. Necropsies were performed 14 to 16 days after treatment and
the reductions (P<0.05) listed below were recorded. No adverse reactions
occurred.

Trial 7685 was conducted in South Africa to confirm the efficacy of ivermectin
against nematodes. Twenty sheep raised worm-free except for Trichuris
spp. were inoculated with infective nematode larvae and randomly allocated
to two groups. Eight control sheep were untreated and the 12 sheep in the other
group were given ivermectin in propylene glycol once orally at 200 mcg/kg when
the nematodes were in the third larval stage. necropsies were performed 34 to
36 days after treatment and the reductions (P<0.05) listed below were
recorded. No adverse reactions occurred.

Trial 7686 was conducted in South Africa to confirm the efficacy of ivermectin
against nematodes. Nineteen sheep were artificially infected with Ostertagia
circumcincta and allocated to two groups. The sheep harbored natural
infections of Trichuris ovis. Seven sheep were untreated controls, and
12 sheep were given ivermectin in propylene glycol once orally at 200 mcg/kg
when the nematodes had reached the fourth larval stage (L4) of development.
Necropsies were performed 38 to 40 days after treatment and the reductions
(P<0.05) listed below were recorded. No adverse reactions occurred.

Trial 7687 was conducted in South Africa to confirm the efficacy of ivermectin
against nematodes. Nineteen sheep were artificially infected and allocated to
two groups. Some sheep also harbored natural infections of Trichuris
spp. Seven sheep were untreated controls, and 12 sheep were given
ivermectin in propylene glycol once orally at 200 mcg/kg when the nematodes
were in the adult stage of development. Necropsies were performed 14 to 16 days
after treatment and the reductions (P<0.05) listed below were recorded. No
adverse reactions occurred.

Trial 7688 was conducted in South Africa to confirm the efficacy of ivermectin
against nematodes. Twenty sheep raised worm-free except of Trichuris
spp. were inoculated with infective nematode and randomly allocated to two
groups. Eight control sheep were untreated and the 12 sheep in the other group
were given ivermectin in micelle solution once orally at 200 mcg/kg when the
nematodes were in the parasitic third stage. Necropsies were performed 41 to 43
days after treatment and the reductions (P<0.05) listed below were recorded.
No adverse reactions occurred, except that one ivermectin-treated sheep died as
a result of pharyngeal trauma inflicted while being dosed.

Trial 7898 was conducted in the United Kingdom to confirm the efficacy of
ivermectin against nematodes. Twelve sheep with natural infections were also
artificially infected with Chabertia ovina and randomly allocated to two groups
of equal size. Control sheep were untreated, and the sheep in the other group
were given ivermectin in micelle solution once orally at 200 mcg/kg.
Necropsies were performed l4 days after treatment and the reductions
(P<0.05) listed below were recorded. Data from 6 other sheep given a second
formulation are irrelevant to this summary and have been deleted. No adverse
reactions
occurred.

Trial 8301 was conducted in the United Kingdom to confirm the efficacy of
ivermectin against nematodes. Twelve sheep were artificially infected and
randomiy allocated to two groups of equal size. Control sheep were untreated,
and sheep in the other group were given ivermectin in micelle solution once
orally at 200 mcg/kg. Necropsies were performed 14 days after treatment and
the reductions (P<0.05) listed below were recorded. Data from 18 other
sheep given other doses and other formulations are irreievant to this summary
and have been deleted. No adverse reactions occurred.

Trial 8421 was conducted in New Zealand to confirm the efficacy
of ivermectin against nematodes. Fourteen sheep with natural
infections were randomly allocated to two groups of equal size.
Control sheep were given vehicle, and the other sheep were given
ivermectin in micelle solution once orally at 200 mcglkg.
Necropsies were performed seven to nine days after treatment and
the reductions (P<0.05) listed below were recorded. No adverse
reactions occurred. Data from 21 other sheep given other drugs
are irrelevant to this summary and have been deleted.

Trial 8451 was conducted in the United States to compare the
efficacy of ivermectin in propyiene glycol or micelle
formulations against adult nematodes. Eighteen naturally
infected sheep were randomly allocated to three groups of equal
size. Sheep in one group were untreated controls, and sheep in
the other two groups received ivermectin at 200 mcg/kg when
formulated in either vehicle. Necropsies were performed seven
days after treatment, and the reductions (P<0.05) listed below were
recorded. Based on the data obtained, ivermectin given in
either formulation was concluded to be equivalent. No adverse
reactions occurred.

Trial 7728 was conducted in the United States as a field evaluation of the
efficacy of Ivermectin administered at use level in the commercial formulation.
Eighty sheep were given ivermectin at 200 mcg/kg and 20 were used as controls.
The incidence of positive nematode egg counts in fecal samples examined from
ivermectin-treated sheep was reduced from 80% before treatment to 5% after
treatment. No adverse reactions occurred.

A series of trials was conducted to demonstrate that ivermectin given in single
oral administrations to sheep at 200 mcg/kg is safe and that the drug has a
wide therapeutic index. These trials involved investigations of tolerance,
toxic syndrome, breeding sheep safety, and other potential problems.

Tolerance

Trial 7524 was conducted in France as a tolerance trial. Fifty sheep (equal
numbers of each sex) about three months old, were allocated to five groups of
equal size. Treatments included vehicle or ivermectin in micelle solution once
orally at 200 mcg/kg, 1000 mcg/kg, 2000 mcg/kg. or 4000 mcg/kg (or 1, 5, 10, or
20 times the recommended therapeutic dose level). Except for the first male
replicate which was treated orally, all other treatments were given via
intraesophageal intubation. Each animal was weighed and observed clinically
during the 21 days after treatment. No adverse effects were observed except
for coughing in the replicate treated orally.
Investigator: J. Foix, D.M.V., 10 Rue Du Mont D'Urville,
78200 Manres La Jolie, France

Toxic Syndrome

Trial 7000 was conducted in the United States as a toxic syndrome trial. Ten
male and ten female sheep (28 to 39 kg in weight) were randomly allocated to
five groups of equal size. Control sheep were given water in a quantity
comparable to the average of all other treatment volumes, and other groups were
given ivermectin in propylene glycol at 300 mcg/kg, 2000 mcg/kg, 4000 mcg/kg,
and 8000 mcg/kg (or 1.5, 10, 20, and 40 times the use level). Two days later
an additional two sheep were each given 315 m1 of propylene glycol alone. All
but one of the ivermectin-treated sheep coughed during or immediately following
treatment and had other signs including head shaking and labia1 licking. Sheep
given ivermectin at 300 mcg/kg and 2000 mcg/kg remained clinically normal
during the trial. Sheep given ivermectin at 4000 mcg/kg were mildly depressed
and ataxic during the first day after treatment. At 8000 mcg/kg, ataxia and
depression were observed and one sheep became comatose. Three days later, all
sheep appeared normal. The two sheep given propyiene glycol had the same signs
as the sheep given ivermectin at 8000 mcg/kg; one died within 24 hours of
treatment and the other recovered. All the test sheep were Killed 21 to 23
days after treatment. but no gross or histologic lesions were present that
could be associated with the administration of ivermectin. In conclusion. the
adverse effects observed in the sheep given ivermectin formulated in propylene
glyco1 seemed to be more attributable to the vehicle than to ivermectin.

Trial 6455 was conducted in New Zealand to determine whether ivermectin has any
teratogenic effects in ewes. There were 364 ewes allocated to seven groups of
equal size. Treatments were given to ewes once during the first, second, or
third week of pregnancy; and again during the fourth, fifth, or sixth week of
pregnancy. Three replicates were given ivermectin once orally at 400 mcg/kg,
three replicates were given vehicle in a comparable volume, and one replicate
was untreated. There was a range of seven days in ages of embryos exposed to
treatment within each regimen, and hence there were ewes treated on each of the
first 43 days of pregnancy. No marked differences in rates or return to estrus
after matings were recorded among the ewes. All lambs born alive were examined
shortly after birth, and those born dead were necropsied. No adverse effects
attributable to treatment with ivermectin were detected either in the ewes or
in their
lambs.

Trial 6660 was conducted in Australia to determine whether ivermectin has any
effects in ewes on pregnancy. Thirty-six ewes were allocated to two groups of
equal size. Ewes in one group were given ivermectin orally at 400 mcg/kg
(twice the use level) at 14-day intervaIs starting before the 51st day of
pregnancy and ending when their ewes were weaned at 56 days of age. The second
group of ewes was given comparable volumes of vehicle on the same schedule. No
significant differences were found between groups in the numbers of lambs
born, birth weight of lambs, or the weaning weights. No stillbirths occurred
in either group and no deformities were found in any lambs. Ivermectin was
concluded to have had no effect on these pregnant ewes or their lambs.

Trial 6729 was conducted in South Africa to determine whether Ivermectin has
any effects in the reproductive performance of rams. Twenty breeding rams were
allocated to two groups of equal size. Rams in one group were untreated
controls whereas the other group of rams was given ivermectin once orally at
400 mcg/kg (twice the use level). Semen samples were collected from each ram
by electro-ejaculation three times before and twice after each treatment, and
no important differences were found between the groups. Further, each ram was
hemicastrated six days after the last observation; histologica1 examination
revealed no differences between the groups. Ivermectin was concluded to have
had no effect on the breeding performance of the rams studied.

There were 124 field trials conducted to assess safety of ivermectin use under
field conditions. A dose of 200 mcg/kg was utilized in one United States tria1
and in another in New Zealand. All the other trials utilized a dose of 400
mcg/kg. propyiene glycol was used as vehicle in 95 trials whereas micelle
vehicle was used in the other 29, including the U.S. trial.

These trials involved 10,730 sheep given ivermectin at 400 mcg/kg, of which
1782 were given ivermectin in micelle vehicle at 400 mcg/kg and 80 given
ivermectin in micelle solution at 200 mcg/kg. No adverse reactions occured in
these trials. However, the amount of coughing in sheep given the propylene
glyco1 vehicle was sufficient to result in a formulation change to the
micelle solution.

During the efficacy trials, no adverse reactions attributable to ivermectin
occured. There was, as already stated, sufficient coughing among sheep given
the propylene glycol formulation to result in the selection of a micelle
vehicle for commercial use. Three deaths occured in the trials, including an
illness existing prior to treatment. Pharyngeal trauma occuring during dosing,
and enterotoxemia. None of these deaths was caused by ivermectin.

Species and strain of Test Animal: Beagle Dogs. Number of Animals of Each
Sex/Group: 2/sex/group. Dose Levels: 2.5, 5.0, 10.0 mg/kg.

Route of Drug Administration: Oral.

Toxicity Observed: There was no mortality. Mydriasis was noted following dosing
of dogs at 2.5 or l0 mg/kg/day. Tremors were seen in dogs given 5 and l0
mg/kg/day, and one dog at 10 mg/kg/day was comatose on the morning following
dosing. This dog recovered within 48 hours of dosing but was ataxic and had
occasional tremors until 72 hours post-dosing.

Toxicity Observed: Increased mortality among offspring in the l.6 mg/kg/day
dosage level group. The purpose of this study was to provide offspring which
had been exposed to MK-933 in utero and throughout the lactation period for use
in a subsequent three-month oral toxicity study in rats (TT #78-037-0).

Species and Strain: Charles River rats born of F0 females which had been
administered MK-933 throughout gestation and lactation in a study previously
described (TT #78-7l0-0).

Number of Animals/Sex/Group: 20

Dose Levels: 0.4, 0.8 and l.6 mg/kg/day daily for 14 weeks.

Route of Drug Administration: Oral.

Parameters Examined: Daily clinical appearance, body weight gain,
opthalmologic, hematologic and serum biochemical studies in Drug Weeks 4, 8 and
13. All rats were necropsied, detailed microscopic examination of tissues from
all control and high dose rats; liver, kidney, spleen and bone marrow from all
rats were also examined microscopically.

Toxicity Observed: Splenomegaly, extramedullary hematopoiesis, and iron
positive pigment in the renal tubular epithelium in three rats from the 1.6
mg/kg/day group and one from the 0.6 mg/kg/day group. These changes suggest
possible drug-related intravascular hemolysis. There were no other ante- or
postmortem changes related to drug administration.

Toxicity Observed: Mydriasis and slight body weight loss at doses of l and 2
mg/kg/day. Four dogs in the 2 mg/kg/day group developed tremors, ataxia.
anorexia and dehydration and were sacrificed prior to scheduled necropsy.
Treatment-related postmortem changes were limited to small areas of agonal
gastrointestinal congestion and/or hemorrhage in 2 of the 4 dogs that were
sacrificed in poor physical condition.

Toxicity Observed: There were treatment-related deaths of 1, 1 and 3 females
in the 0.2, 0.4 and 0.8 mg/kg/day dosage groups. respectively. In most of
these mice intermittent whole body muscular tremors were observed 1 to
2 days prior to death or sacrifice. Teratogenicity was evident at external
examination of fetuses in the 0.8 and 0.4 mg/kg/day dosage level groups as an
increased incidence of cleft palate.

Toxicity Observed: In the 10 mg/kg/day dosage level, group 3 females were
sacrificed in poor condition. Treatment-related clinical signs of toxicity in
this dosage group included sedation and focal alopecia. Teratogenicity, as
evidenced by cleft palate was observed at a dose of lO mg/kg/day.

Toxicity Observed: Slight to marked sedation. significant decrease in mean
body weight gain, and an increased number of abortions occurred in females from
the 6 mg/kg/day group. Fetotoxicity was evident in the mg/kg/day group as a
significant increase in fetal deaths and a decrease in mean live fatal weight,
Decrease in mean live fetal weight was also apparent in fatuses from the
mg/kg/day group. Teratogenicity was evident as a dose-related incidence of
cleft palate and clubbed forepaws among letuses from the 3 and 6 mg/kg/day
dosage groups.

Dose Levels and Duration of Treatment: 0.4, 1.2 and 3.6 mg/kg/day; the study
was designed so that dosing of F0, F1b and F2b weanling male and female rats
would be continuous for 70 days and throughout the production of two litters in
each of three generations. The study was terminated prior to mating of the F1b
generation for production of an F2b litter when it became apparent that
toxicity observed in F1a, F1b and F2a offspring would preclude the
establishment of a no-effect level.

Toxicity Observed: There was a decrease in survival of F1a offspring in the
3.6 mg/kg/day groups. and in F1b offspring from the 1.2 and 0.4 mg/kg/day
dosage levels. Surviving pups in all treatment groups showed decreased weight
gain during the lactation period.

Dose Levels and Duration of Treatment: 0.05, 0.1, 0.2 and 0.4 mg/kg/day;
dosing of F0 male and female rats was continuous for 71 days and continued
throughout the production of two litters in each of three successive
generations.

Dose Levels and Duration of Treatment: 2.5
mg/kg/day; one group received tritium-labeled MK-932 as a solution in sesame
oil for days, then throughout mating, gestation and lactation until Day 9
postpartum. A second group received tritium-labeled MK-932 at the same dosage
level from Days 1 to 9 postpartum.

Route of Drug Administration: Oral.

Parameters Examined: Maternal plasma, milk, as well as samples of blood,
brain, liver and carcass from mother and offspring were analyzed for drug
residues.

Results: Steady state plasma drug levels were reached in the maternal animal
within 10 days of dosing. Maternal drug plasma levels increased three- to
four-fold on Day l postpartum, possibly indicating a mobilization of depot fat
during the latter stages of gestation. Concentrations of MK-932 in the milk
were three to four times higher than revels in the plasma. based on estimates
of milk intake by neonatal rats, the quantity of MK-932 secreted in the milk
reached levels as high as 50% of the LD50 for infant rats. Neonatal blood/brain
ratios indicated that the blood/brain barrier was formed in the rat between Day
6 and Day lO postpartum. The results of this study indicated that the high concentrations of MK-932 in the
milk of 1aerating dams chronically administered the drug may explain the
toxicity observed among offspring in MK-933 multigeneration studies during the
lactation period.

No Observed Effect Level: Not applicable.

B. Safe Concentration of Residues

The lowest no-observable-effect-levels
(NOEL) in the battery of toxicity studies described in Section A above were
determined in the multigeneration study in rats (0.1 mg/kg/day) and in the oral
teratogenic study in the mouse (0.1 mg/kg/day for maternotoxicity and 0.2
mg/kg/ day for teratogenicity). The minimum safety margins required for the
effects observed in these studies are 100x for the multigeneration study and
for the maternotoxicity in the mouse teratological study and 1000x for the
teratological effects also seen in the latter study. Due to the significance
of the terata (cleft palate) seen in the teratology study the 1000x safety
factor was used for determining an acceptable daily intake of up to two tenths
micrograms (0.2) per kg of ivermectin residue by an individual in food.

i.e.. 0.2 mg + 1000 safety factor = 0.2 mcg

A safe level in the muscle tissues of swine is calculated from the acceptable
daily intake. assuming the average weight of man to be 60 kg and the daily
human intake of muscle to be 500 g, as follows:

safe concentration in muscle = (60 kg) (0.2 mcg/kg) = 24 ppb.
500

When rounded to the nearest 5 ppb the safe concentration in muscle then becomes
25 ppb. The safe concentration of residues in liver, kidney and fat are
determined from this number using appropriate food consumption values (food
factor) for these tissues. Therefore, the safe concentrations are:

The H2B1a component of the unaltered drug has been selected as the marker
substance for ivermectin, since it is the major component in liver at all
slaughter periods and has the appropriate depletion characteristics in that
tissue. An Rm value of 30 ppb has been established for liver (the target
tissue). since
H2Bla represents 25% of the residue in liver when the total residue in the
tissue is at the safe concentration of 125 ppb.

C. Metabolism and Total Residue Depletion Studies

Total radioactive residue (averaged value from three animals) from two
separate experiments in edible tissues of sheep dosed intraruminallY with
3H-labeled MK-933 at levels of 0.3 mg/kg body weight is shown in the table
below:

Although both liver and fat were candidate target tissues. liver has been
chosen as the target tissue because of the integrity of that tissue as one
organ and the difficulty in extracting and isolating residues of the drug from
fat. The H2B1a component of the unaltered drug has been selected as the
marker substance for ivermectin, since it is the major component in liver at
all slaughter periods and has the appropriate depletion characteristics in that
tissue. An Rm of 30 ppb has been established for liver (the target tissue).

That value represents the average 1eve1 of the H2B1a marker in liver tissue as
measured by the regulatory assay when the average total residue in fat (the
tissue in which residues persist longest) depletes to the safe concentration of
125 ppb.

The radioactive residue in the edible tissues is essentially all extractable in
organic solvents indicating that there is very little, if any, intractable
covalently bound residues in these tissues. The unaltered drug (H2B1a and
H2B1b) accounts for about 78% of the total radioactive residue in liver at 3
days and about 65% at 5 days after dosing. In fat, the unaltered drug accounts
for about 56% of the total residue 3 days after dosing and 33% at 5 days after
dosing.

The major polar metabolite in the sheep liver is identified to be
24-hydroxymethyl-H2B1a. Other polar
metabolites are identified as the monosaccharide of 24-hydroxymethyl-H2B1a and
the 24-hydroxymethyl-H2Blb. The total residue identified as unaltered drug or known metabolites is
68% in the liver at 5 days after dosing. In fat, the major metabolites are
nonpolar. Chemical and enzymatic hydrolysis studies suggest that these
metabolites exist in the fat as an acy1 esters of the 24-hydroxymethylated
metabolite. None of the unidentified metabolites in tissues represented more
than 10% of the total residue.

Since the unaltered drug is the major component in the liver at all slaughter
periods, the unaltered drug (i.e., H2B1a component) should be a satisfactory
marker substance.

Comparative metabolism studies indicate a striking similarity in metabolism of
ivermectin in sheep and rat, the test species. The analyses for unaltered drug
in various tissues, the HPLC profile of the radioactive residue in the liver,
and the in vitro liver microsomal metabolism are all comparable in sheep and
rats. Thus, the test species is exposed to the major drug residue components
(i.e., unaltered drugs and known metabolites) known to be present in sheep
tissues.

A study was performed to determine residues in sheep tissues resulting from
dosing the animals with ivermectin orally with a micellar formulation at 0.3
mg/kg.
The vehicle contained 0.8 mg of ivermectin/ml in an aqueous vehicle containing
20% propylene glycol and B% surfacant. Three wethers and two ewes were
sacrificed at each withdrawal time. The withdrawal times involved being 1, 3,
5, 7, 10, and 14 days. An additional set of five animals served as
controls.

Liquid chromatographic-fluorescence determinative tissue residue assays were
run on all Livers (the target tissue) from this study. Average residues found
were as follows:

Day post dose 1 3 5 7 10 14 Control
ppb found 72 12 11 8 0 0 0

The analytical method used to make the determination quoted easily has a lower
limit of reliable measurement of 10 ppb. The 1imit of detection is 1-2 ppb.
The Rm value for sheep derived from toxicity and metabolism data has been
determined to be 30 ppb. Statistical analysis of the depletion data using the
upper tolerance limit containing 99 percentile of the population with
confidence yields a withdrawal period of 11 days.

E. Regulatory Methods

Ivermectin Determinative Assay Scheme

The determinative assay measures the marker substance, 22,23-dihydroavermectin
B1a, by liquid chromatography
of a fluorescent derivative. The marker substance is extracted into isooctane
from an aqueous acetone homogenate of the liver tissue. The isooctane is
removed by evaporation and the extract purified by a series of
acetonitrile-hexane-water distributions. The fluorescent derivative is formed by heating with an acetic anhydride/methylimidazole
reagent. A chloroform solution is purified over a silica column and
concentrated by evaporation/reverse phase liquid chromatography is carried out
using 5:95 water/methanol and fluorescence detection. Quantitation is obtained
using a standard curve for the marker substance carried through the
derivatization and subsequent steps. Recoveries are in the range of 10-88% and
Lm is estimated to be 10 ppb with a limit of detection of 1-2 ppb.

Ivermectin Confirmatory Scheme

The sample preparation and purification steps of the confirmatory assay are
essentially the same as the determinative assay. The specificity is obtained
by the production of two new species just prior to derivatization. The new
species are produced by removing one of the saccharide groups with 15 sulfuric
acid in isopropanol to form the monosaccharide or removing both saccharide
groups with 15 sulfuric acid in methanol to form the aglycone of
22,23-dihydroavermectin B1a. Since these two treatments are so similar the
formation of the two new species and their chromatographic properties is unique
and hence confirmation of the presence of 22,23-dihydroavermectin
B1a.

In the actual test, the sample is split in three parts. One part is used for
each of the sulfuric acid treatments. These samples are separated from the
sulfuric acid by extractions and fluorescing derivatives of the two new
compounds are made. The third aliquot is derivatized without pretreatment.
All three derivatives are then extracted into hexane with a small amount of
iso-butyl aicohol present. The liquid chromatographic determination is made as in
the determinative assay.

Three separate peaks are observed at separate retention times which are
compared to standards run through the procedure from the point of adding the
sulfuric acid onward. Presence of and quantitation of the three peaks is
confirmation that ivermectin is present.

Validation

The determinative and confirmatory methods were validated satisfactorily by FDA
and USDA laboratories. The validated regulatory analytical methods for
detection of residues of ivermectin are filed in the Food Additives Manual on
display in FDA's Freedom of Information Public Room (Room 12A-30, 5600 Fishers
Lane, Rockville, MD 20857).

7. Agency Conclusions

The data submitted in support of this NADA ccmply with the requirements of
Section 512 of the Act and demonstrate that Ivomec (Ivermectin) Sheep Drench,
0.08% Solution when used under its proposed conditions of use is safe and
effective.

The requirements under 21 CFR 514.1(b)(3) have been met. The firm has
described practicable methods for determining the quantity of the drug and any
substance formed in food because of its use. Both determinative and
confirmatory methods were validated by FDA and USDA laboratories.

The drug and its metabolites were accounted for by appropriate metabolism
and total residue depletion studies utilizing an acceptable radiotracer.
These studies included comparative metabolism studies in the target (sheep)
and test (rat) species.

A tolerance (Rm of 30 ppb) has been established for the marker residue
(22,23-dihydroavermecrin B1a) in the target tissue (liver) and a withdrawal
period (11 days) has been determined experimentally using the 99% statistical
tolerance limit with 95% confidence under field conditions to assure that the
prescribed use of the subject drug will be safe and that residues of ivermectin
in sheep will be safe for human consumption.

Based on the provisions in the Threshold Assessment guideline, the agency
initially placed ivermectin in toxicity category "C". Following an evaluation
of data from a battery of three short-term geno-toxicity tests and from 90-day
rat studies in the rat and dog which raised no suspicion of carcincgenicity,
the agency assigned ivermectin to category "A". Additional toxicity data
provided by the sponsor were adequate to satisfy the agency's general food
safety requirements for NADA approval.

The Agency concludes that adequate directions for use by the lay persons have
been written for the proposed over-the-counter use of this anthelmintic and
endoparasitic drug which is indicated for the treatment of endoparasites
commonly occuring in sheep.